Method for producing L-amino acid using bacteria belonging to the genus Escherichia

ABSTRACT

A method for producing L-threonine, L-valine, L-proline, L-leucine, L-methionine and L-arginine is provided using  Escherichia  bacteria wherein the L-amino acid productivity of the bacteria is enhanced by increasing the activity of proteins encoded by the b2682 and b2683 genes, or proteins encoded by the b1242 or b3434 gene.

This application is a divisional application under 35 U.S.C. §120 ofU.S. patent application Ser. No. 10/073,293, filed Feb. 13, 2002, issuedas U.S. Pat. No. 7,476,531 on Jan. 13, 2009, and claims priority under35 U.S.C. §119 to Russian Patent Application No. 2001103865, filed Feb.13, 2001, Russian Patent Application No. 2001104998, filed Feb. 26,2001, Russian Patent Application No. 2001104999, filed Feb. 26, 2001,Russian Patent Application No. 2001117632, filed Jun. 28, 2001, RussianPatent Application No. 2001117633, filed Jun. 28, 2001, all of which areincorporated by reference. The Sequence Listing filed electronicallyherewith is also hereby incorporated by reference in its entirety (FileName: US-145D1_Seq_List_Copy_(—)1; File Size: 21 KB; Date Created: May14, 2008).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to biotechnology, and specifically to amethod for producing L-amino acids by fermentation, and morespecifically to genes derived from Escherichia coli bacteria. Thesegenes are useful for improving production of L-amino acids, for example,L-threonine, L-valine, L-proline, L-leucine, L-methionine, andL-arginine.

2. Brief Description of the Related Art

Conventionally, L-amino acids are industrially produced by fermentationmethods utilizing microorganisms obtained from natural sources ormutants which have been specifically modified to enhance production ofthe L-amino acids.

Many techniques designed to enhance L-amino acid production have beendisclosed, for example, transforming microorganisms with recombinant DNA(see, for example, U.S. Pat. No. 4,278,765). These techniques are basedon increasing the activities of the enzymes involved in amino acidbiosynthesis and/or desensitizing the enzymes involved in the feedbackinhibition by the target L-amino acid (see, for example, JapaneseLaid-open application No 56-18596 (1981), WO 95/16042, U.S. Pat. No.5,661,012 or 6,040,160).

Alternatively, the secretion of the target L-amino acid can be increasedwhich can enhance the productivity of the strain producing the L-aminoacid. Bacteria belonging to the genus Corynebacterium in whichexpression of an L-lysine secretion gene is increased (lysE gene) havebeen disclosed (WO 9723597A2). In addition, genes coding for the effluxproteins which act to enhance secretion of L-cysteine, L-cystine,N-acetylserine, or thiazolidine derivatives are also disclosed (U.S.Pat. No. 5,972,663).

At present, several Escherichia coli genes coding for putative membraneproteins which act to enhance L-amino acid production have beendisclosed. The presence of additional copies of the rhtB gene makesbacteria more resistant to L-homoserine and enhances production ofL-homoserine, L-threonine, L-alanine, L-valine and L-isoleucine(European patent application EP994190A2). The presence of additionalcopies of the rhtC gene makes bacteria more resistant to L-homoserineand L-threonine, and enhances production of L-homoserine, L-threonineand L-leucine (European patent application EP1013765A1). The presence ofadditional copies of the yahN, yeaS, yfiK, and yggA genes enhanceproduction of L-glutamic acid, L-lysine, L-threonine L-alanine,L-histidine, L-proline, L-arginine, L-valine, and L-isoleucine (Europeanpatent application EP1016710A2). Even though the complete genomesequence of Escherichia coli strain K-12 has been disclosed (Blattner F.R., Plunkett G., Bloch C. A. et al., Science, 227, 1453-1474, 1997), thefunctions of many ORFs remains unknown.

SUMMARY OF THE INVENTION

An aspect of present invention is to enhance the productivity of L-aminoacid producing microorganism strains and to provide a method forproducing L-amino acids, for example, L-threonine, L-valine, L-proline,L-leucine, L-methionine, and L-arginine, using these strains.

This was achieved by identifying genes coding for proteins which are notinvolved in the biosynthetic pathways of target L-amino acids, but whichenhance production of the target amino acids. An example of such aprotein is a membrane protein having L-amino acid excretion activity.Based on the analysis of the complete genome sequence of Escherichiacoli, proteins with 4 or more putative transmembrane segments (TMS) wereselected. As a result, several genes were identified, specifically,b2682, b2683, b1242 and b3434, and studied. The b2682 and b2683 genesare known as putative CDS genes which encode proteins with unknownfunctions (nucleotide numbers 92 to 829 and 819 to 1154 in GenBankaccession AE000353 U00096, respectively). The b2683 gene is also knownas ygaH. The b1242 gene is known as a putative CDS which encodes aprotein with unknown function (numbers 8432 to 9079 in GenBank accessionAE000222 U00096). The b1242 gene is also known as ychE. The b3434 genealso is known as a putative CDS which encodes a protein of unknownfunction (numbers 1463 to 2056 in GenBank accession AE000420 U00096).The b3434 gene is also known as yhgN.

Also, it has been found that by enhancing the activities of the proteinsencoded by the b2682, b2683, b1242, and b3434 genes, the productivity ofan L-amino acid producing strain is enhanced.

It is an aspect of the present invention to provide an L-amino acidproducing Escherichia bacterium, wherein the bacterium has been modifiedso that the L-amino acid production by the bacterium is enhanced byenhancing activities of proteins selected from the group consisting of:

(A) a protein comprising the amino acid sequence shown in SEQ ID NO:4,

(B) a protein comprising the amino acid sequence shown in SEQ ID NO:4,except it includes deletions, substitutions, insertions or additions ofone or several amino acids, and which has an activity of making thebacterium have enhanced resistance to the L-amino acids and/or theiranalogs.

(C) a protein comprising the amino acid sequence shown in SEQ ID NO:6,

(D) a protein comprising the amino acid sequence shown in SEQ ID NO:6,except it includes deletions, substitutions, insertions, or additions ofone or several amino acids, and which has an activity of making thebacterium have enhanced resistance to the L-amino acids and/or theiranalogs.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the activities of the proteins defined as(A), (B), (C), or (D) are enhanced by either transformation of thebacterium with a DNA coding for the proteins defined as (A), (B), (C),or (D), or by alteration of expression regulatory sequence of the DNA onthe chromosome of the bacterium.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the transformation is performed with amulticopy vector.

It is a further aspect of the present invention to provide a method forproducing an L-amino acid comprising cultivating the bacterium asdescribed above in a culture medium and collecting the L-amino acid fromthe culture medium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-threonine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of the threonine operon.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-valine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of the ilv operon.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-proline.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of genes which encode proteins activein proline biosynthesis.

It is a further aspect of the present invention to provide the methoddescribed above, wherein the L-amino acid is L-leucine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of the leu operon.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-methionine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of the met regulon.

It is a further aspect of the present invention to provide anEscherichia L-amino acid producing bacterium, wherein the bacterium hasbeen modified so that the L-amino acid production by the bacterium isenhanced by enhancing activities of proteins selected from the groupconsisting of:

(E) a protein comprising the amino acid sequence shown in SEQ ID NO:12,

(F) a protein comprising the amino acid sequence shown in SEQ ID NO: 12,except it includes deletions, substitutions, insertions, or additions ofone or several amino acids, and which has an activity of making thebacterium have enhanced resistance to the L-amino acids and/or theiranalogs,

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the activities of the proteins defined as(E) or (F) are enhanced either by transformation of the bacterium with aDNA coding for the proteins defined as (E) or (F), or by alteration ofan expression regulation sequence of the DNA on the chromosome of thebacterium.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the transformation is performed with amulticopy vector.

It is a further aspect of the present invention to provide a method forproducing an L-amino acid comprising cultivating the bacterium asdescribed above in a culture medium and collecting the L-amino acid fromthe culture medium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-threonine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of the threonine operon.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-valine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of the ilv operon.

It is a further aspect of the present invention to provide an L-aminoacid producing bacterium belonging to the genus Escherichia, wherein thebacterium has been modified so that the L-amino acid production by thebacterium is enhanced by enhancing activities of proteins selected fromthe group consisting of:

(G) a protein comprising the amino acid sequence shown in SEQ ID NO:16,

(H) a protein comprising the amino acid shown in SEQ ID NO:16, except itincludes deletions, substitutions, insertions, or additions of one orseveral amino acids, and which has an activity of making the bacteriumhave enhanced resistance to the L-amino acids and/or its analogs, suchas DL-o-methylserine, 6-diazo-5-oxo-L-norleucine andDL-β-hydroxy-norvaline, and having enhanced sensitivity toS-(2-aminoethyl)cysteine,

It is a further aspect of the invention to provide the bacterium asdescribed above, wherein the activities of the proteins defined as (G)or (H) are enhanced by either transformation of the bacterium with a DNAcoding for the proteins defined as (G) or (H), or by alteration of anexpression regulation sequence of the DNA on the chromosome of thebacterium.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the transformation is performed with amulticopy vector.

It is a further aspect of the present invention to provide a method forproducing an L-amino acid comprising cultivating the bacterium asdescribed in a culture medium and collecting the L-amino acid from theculture medium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-arginine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of the arginine regulon.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the L-amino acid is L-proline.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium has been modified so that thebacterium has enhanced expression of genes encoding proteins active inproline biosynthesis.

The method for producing an L-amino acid includes the production ofL-threonine, L-valine, L-proline, L-leucine, L-methionine using theappropriate L-amino acid producing bacterium wherein the activities ofthe proteins comprising the amino acid sequences shown in SEQ ID NO:4and SEQ ID NO:6 are enhanced.

Furthermore, the method for producing an L-amino acid includesproduction of L-threonine using an L-threonine producing bacteriumwherein the activity of the protein comprising the amino acid sequenceshown in SEQ ID NO:12 is enhanced. Also, a method for producing anL-amino acid includes production of L-valine using L-valine producingbacterium wherein the activity of the protein comprising the amino acidsequence shown in SEQ ID NO:12 is enhanced.

Still further, the method for producing an L-amino acid includes theproduction of L-arginine using an L-arginine producing bacterium whereinthe activity of the protein comprising the amino acid sequence shown inSEQ ID NO:16 is enhanced. Also, the method for producing an L-amino acidincludes the production of L-proline using an L-proline producingbacterium wherein activities of the comprising amino acid sequence shownin SEQ ID NO:16 are enhanced.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the construction of plasmid pΔlacZ.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be explained in detail below.

The bacterium of the present invention is an L-amino acid producingbacterium belonging to the genus Escherichia, wherein the bacterium hasbeen modified so that the L-amino acid production by the bacterium isenhanced by increasing the activities of the proteins described hereinin the bacterium.

“L-amino acid producing bacterium” means a bacterium which has anability to cause accumulation, that is, produce an L-amino acid when thebacterium is cultured in the medium. The L-amino acid producing abilitymay be an inherent property of the wild-type bacterium or may beimparted or enhanced by breeding.

The proteins which have increased activity in the bacterium are proteinswhich increase the production of a target L-amino acid. Specifically,the bacterium is an L-amino acid producing bacterium belonging to thegenus Escherichia which has enhanced activity of at least one or two ofthese proteins.

The phrase “enhancing or increasing an activity of a protein” means thatthe activity per cell is higher than that in a non-modified strain, forexample, a wild-type Escherichia bacterium. For example, the activity ofa protein is increased typically when the number of the proteinmolecules increases per cell, or when the specific activity per theprotein molecule increases, and so forth. Furthermore, a wild-typeEscherichia bacterium may be used for comparison, that is, as a control,and specifically, a wild-type strain of Escherichia coli.

Specifically, an example of the bacterium is one which harbors DNA whichoverexpresses either the b2682 or b2683 gene, and preferably both ofthese genes, on the chromosomal DNA or on a plasmid in the bacterium. Asa result, the bacterium has an enhanced ability to produce an L-aminoacid, for example, L-threonine, L-valine, L-proline, L-leucine, and/orL-methionine. Another example of the bacterium is one which harbors DNAwhich overexpresses the b1242 gene on the chromosomal DNA or on aplasmid in the bacterium, and has an enhanced ability to produce anL-amino acid, for example, L-threonine and/or L-valine. A third exampleof the bacterium is one which harbors DNA which overexpresses the b3434gene on the chromosomal DNA or on a plasmid in the bacterium, and has anenhanced ability to produce an L-amino acid, for example, L-arginineand/or L-proline.

The proteins overexpressed by the genes as described above include thoseshown in SEQ ID NOs: 4 and 6, as well as variants of these proteins. Thevariants include proteins having the amino acid sequences of SEQ ID NOs.4 and 6, but which include one or more deletions, substitutions,insertions, or additions of one or several amino acids, and which havean activity of making the bacterium have enhanced resistance to theL-amino acids and/or their analogs.

The number of amino acids which can be changed differs depending on theposition or the type of amino acid residue in the three-dimensionalstructure of the protein. It may be 1 to 24, preferably 1 to 12, andmore preferably 1 to 5 for the protein having the amino acid sequence ofSEQ ID NO:4, and 1 to 11, preferably 1 to 7, and more preferably 1 to 5for the protein having the amino acid sequence of SEQ ID NO:6.

The proteins overexpressed by the genes described above also includethose proteins having the amino acid sequence shown in SEQ ID NO:12 andvariants thereof. The variants include proteins having the amino acidsequence of SEQ ID NO: 12 but which include one or more deletions,substitutions, insertions, or additions of one or several amino acids,and which have an activity of making the bacterium have enhancedresistance to the L-amino acids and/or their analogs.

The number of amino acids which may be changed differs depending on theposition or the type of amino acid residue in the three-dimensionalstructure of the protein. For the protein of SEQ ID No:12, it may be 1to 22, preferably 1 to 11, and more preferably 1 to 5.

Also, the proteins encoded by the genes described above include thosehaving the amino acid sequence shown in SEQ ID NO. 16 and variantsthereof. The variants include proteins having the amino acid sequence ofSEQ ID NO: 16, but which include one or more deletions, substitutions,insertions, or additions of one or several amino acids, and which havean activity of making the bacterium have enhanced resistance to theL-amino acids and/or their analogs, such as DL-o-methylserine,6-diazo-5-oxo-L-norleucine and DL-β-hydroxy-norvaline, and havingenhanced sensitivity to S-(2-aminoethyl)cysteine.

Again, the number of amino acids which may be changed differs dependingon the position or the type of amino acid residue in thethree-dimensional structure of the protein. For the protein having theamino acid sequence of SEQ ID NO:16, it may be 1 to 20, preferably 1 to10, and more preferably 1 to 5.

Enhanced resistance to L-amino acids and/or their analogs means thebacteria with this enhanced resistance has the ability to grow on aminimal medium containing the L-amino acid or its analog at aconcentration which the unmodified strain or the wild-type strain, orthe parental strain of the bacterium cannot grow. It can also mean thatthe bacterium has the ability to grow faster on a medium containing theL-amino acid or its analog than the unmodified strain or the wild-typestrain, or the parental strain of the bacterium.

More specifically, the E. coli strain has enhanced resistance to theL-amino acid or its analog if the strain forms a colony which is largerthan that of the unmodified strain or wild-type strain of E. coli after2-4 days incubation at 37° C. on a plate with solid Adams medium at 37°C. when the strain is cultivated on an agar medium containing theL-amino acid or its analog under appropriate growth conditions.Appropriate growth conditions refer to temperature, pH, air supply, orthe optional presence of essential nutrients or the like for the chosenE. coli strain.

Examples of L-amino acid analogs include, but are not limited to,3,4-dihydroproline, DL-thiaisoleucine, DL-o-methylserine, 4-azaleucine,norleucine, L-o-fluorophenylalanine, DL-o-fluorophenylalanine,homoserine, 6-diazo-5-oxo-L-norleucine, and DL-β-hydroxy-norvaline.

The concentration of an L-amino acid or its analog which inhibits thegrowth of the unmodified strain or the wild-type strain of the bacteriumvaries significantly, for example, from 0.5 μg/ml for DL-thiaisoleucineto 9600 μg/ml for DL-o-methylserine, depending on the structure of thechosen compound. For example, this concentration is generally 7 to 70μg/ml, preferably 20 to 25 μg/ml for 3,4-dihydroproline; generally 0.5to 5 μg/ml, preferably 0.9 to 1.1 for DL-thiaisoleucine; generally 1100to 9600 μg/ml, preferably 3000 to 3500 for DL-o-methylserine; generally15 to 150 μg/ml, preferably 40 to 50 μg/ml for 4-azaleucine; generally150 to 1500 μg/ml, preferably 450 to 550 μg/ml for norleucine; generally0.6 to 6 μg/ml, preferably 1.5 to 2 μg/ml for L-o-fluorophenylalanine;generally 2 to 20 μg/ml, preferably 5 to 7 μg/ml forDL-o-fluorophenylalanine; and generally 330 to 3300 μg/ml, preferably900 to 1100 μg/ml for homoserine, generally 5 to 50 μg/ml, preferably 12to 18 for 6-diazo-5-oxo-L-norleucine, and generally 25 to 250 μg/ml,preferably 70 to 90 μg/ml for DL-β-hydroxy-norvaline

Sensitivity to L-amino acids and/or their analogs means that thebacteria are able to grow for longer proliferation times as compared tothe unmodified strain or the wild-type strain on a minimal mediumcontaining the L-amino acid or its analog. Alternatively, sensitivity toL-amino acids and/or their analogs means that the bacteria is unable togrow on a minimal medium containing the L-amino acid or its analog atthe same concentration that the unmodified strain or the wild-typestrain is able to grow. An example of an L-amino acid analog isS-(2-aminoethyl)cysteine. The concentration is generally 0.2 to 2.0μg/ml, preferably 0.5 to 1.0 μg/ml for S-(2-aminoethyl)cysteine.

The activities of the above proteins in the bacterium can also beenhanced by transforming the bacterium with DNA coding for the proteinas described above, or by altering an expression regulatory sequence ofthe DNA on the chromosome of the bacterium.

The DNA which is used to modify the bacterium codes for a putativemembrane protein. More specifically, the DNA codes for a protein whichhas 4 or more transmembrane segments. This DNA may code for proteinswhich have L-amino acid excretion activity. More concretely, the DNA isthe b2682, b2683, b1242, and b3434 genes. The coding region of the b2682gene at positions 728-738 and the coding region of the b2683 gene atpositions 1-11 are overlapping. Therefore, both genes can be obtainedby, for example, PCR using primers having the nucleotide sequence shownin SEQ ID Nos: 1 and 2 as a single PCR product. The b1242 gene can beobtained by, for example, PCR using primers having the nucleotidesequence shown in SEQ ID No: 9 and 10. The b3434 gene can be obtainedby, for example, PCR using primers having the nucleotide sequence shownin SEQ ID No: 13 and 14.

Analysis of the complete genome sequence of Escherichia coli has allowedfor selection of the genes coding for proteins having 4 or more putativeTMS. Proteins with known function and transporters described by PaulsenI. T., Sliwinski M. I., Saier M. H. (J. Mol. Biol., 1998, 277, 573) andLinton K. J., Higgins C. F. (Molecular Microbiology, 1998, 28(1), 5)were excluded from the group to be screened. As a result of diligentscreening among the rest of the genes, several genes coding for putativemembrane exporters were chosen. As a result, it was found that theoverexpression of the b2682 and b2683 genes, or the b1242 or b3434 genesenhances the L-amino acid production in an L-amino acid producingstrain.

The DNA of the present invention includes a DNA coding for the proteinwhich includes one or more deletions, substitutions, insertions, oradditions of one or several amino acids in one or more positions in theproteins having the amino acid sequences of SEQ ID NOs. 4 and 6, as longas the activity of the protein is not lost. Although the number of aminoacids which can differ depends on the position or the type of the aminoacid residue in the three-dimensional structure of the protein, it maybe 1 to 24, preferably 1 to 12, and more preferably 1 to 5 for theprotein having the amino acid sequence of SEQ ID NO.4, and 1 to 11,preferably 1 to 7, and more preferably 1 to 5 for the protein having theamino acid sequence of SEQ ID NO:6.

Furthermore, the DNA of the present invention includes DNA coding forproteins which include one or more deletions, substitutions, insertions,or additions of one or several amino acids in one or more positions inthe protein having the amino acid sequence of SEQ ID NOs: 12 and 16 aslong as the activity of the protein is not lost. Although the number ofthe amino acids which can differ depends on the position or the type ofthe amino acid residues in the three-dimensional structure of theprotein, it may be 1 to 22, preferably 1 to 11, and more preferably 1 to5 for the protein having the amino acid sequence of SEQ ID NO: 12, and 1to 20, preferably 1 to 10, and more preferably 1 to 5 for the protein ofSEQ ID NO: 16. The DNA coding for the protein variants of the proteinsshown in SEQ ID NOs: 4, 6, 12, and 16 may be obtained by, for example,modification of the nucleotide sequence using site-directed mutagenesisso that one or more amino acid residues is deleted, substituted,inserted, or added. The modified DNA can be obtained by conventionalmethods, for example by treating with reagents and under conditionswhich result in the generation of mutations. Such reagents includehydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, and nitrous acid,and conditions which result in the generation of mutants include thetreatment of the bacterium harboring the DNA with UV irradiation.

The DNA of the present invention includes variants which can be found inthe different strains and variants of bacteria belonging to the genusEscherichia due to natural diversity. Such variant DNA can be obtainedby isolating DNA which hybridizes with the b2862, b2683, b1242, or b3434genes or a part of the genes under the stringent conditions, and whichcodes for a protein which enhances L-amino acid production. The term“stringent conditions” refers to conditions under which a so-calledspecific hybrid is formed, and non-specific hybrid is not formed. Forexample, stringent conditions includes conditions under which DNAshaving high homology, for instance DNAs having homology no less than 70%to each other, are hybridized. Alternatively, the stringent conditionsare exemplified by the typical conditions used for washing in Southernhybridization, e.g., 60° C., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1%SDS. A partial sequence of the nucleotide sequence of SEQ ID NO: 3 orSEQ ID NO: 5 can be used as a probe for the variants which hybridizes toany of the b2862, b2683, b1242, or b3434 genes. Such a probe may beprepared by PCR using oligonucleotides produced based on the nucleotidesequence of SEQ ID NO: 3, 5, 11 or 15 as primers, and a DNA fragmentcontaining the nucleotide sequence of SEQ ID NO: 3, 5, 11 or 15 as atemplate. When a DNA fragment of about 300 bp is used as the probe, theconditions of washing for the hybridization are, for example, 50° C.,2×SSC, and 0.1% SDS.

Transformation of a bacterium with DNA coding for a protein meansintroduction of the DNA into bacterial cell, for example, byconventional methods, which results in increasing expression of the genecoding for the protein and enhancing the activity of the protein in thebacterial cell.

Gene expression can be enhanced by increasing the gene copy number, forexample. Also, introducing a gene into a vector that is able to functionin the chosen Escherichia bacterium increases the copy number of thegene. Multi-copy vectors such as pBR322, pMW119, pUC19, pET22b or thelike, are preferably used for this purpose.

Gene expression can also be enhanced by introducing multiple copies ofthe gene onto the bacterial chromosome by, for example, homologousrecombination or the like.

When it is desired to enhance the expression of two or more genes, thegenes may be located on the same plasmid, or on different plasmids. Itis also acceptable that one of the genes is located on a chromosome, andthe other gene is located on a plasmid.

Alternatively, gene expression can be enhanced by altering an expressionregulatory sequence of the gene, such as by introducing a mutation in aninherent, i.e. native, expression regulatory sequence of the gene suchas a promoter so that the expression of the gene is enhanced(WO00/18935), and/or placing the DNA under the control of a potentpromoter. For example, the lac promoter, trp promoter, trc promoter, andP_(L) promoter of lambda phage are all known as potent promoters. Theuse of a potent promoter can be combined with increasing the copy numberof the gene.

The bacterium of the present invention can be obtained by introducingthe aforementioned DNAs into a bacterium which inherently has theability to produce L-amino acids, or the ability to produce L-aminoacids can be imparted to the bacterium. Parent strains for deriving thebacteria of the present invention include L-threonine producingEscherichia bacteria such as VL2054 (VKPM B-8067), VNIIGenetika 472T23(U.S. Pat. No. 5,631,157), VKPM B-3996 (U.S. Pat. Nos. 5,175,107 and5,976,843), KCCM-10132 (WO009660A1), KCCM-10133 (WO009661A1) or thelike; L-valine producing Escherichia bacteria such as H-81 (VKPMB-8066), NRRL B-12287 and NRRL B-12288 (U.S. Pat. No. 4,391,907), VKPMB-4411 (U.S. Pat. No. 5,658,766), VKPM B-7707 (European patentapplication EP1016710A2) or the like; L-proline producing Escherichiabacteria such as NRRL B-12403 and NRRL B-12404 (GB2075056), VKPM B-8012(Russian patent application 2000124295), plasmid mutants described inthe patent DE3127361, plasmid mutants described by Bloom F. R. et al.(The 15^(th) Miami winter symposium, 1983, p. 34) or the like; L-leucineproducing Escherichia bacteria such as H-9070 (FERM BP-4704) and H-9072(FERM BP-4706) (U.S. Pat. No. 5,744,331), VKPM B-7386 and VKPM B-7388(RU2140450), W1485atpA401/pMWdAR6, W1485lip2/pMWdAR6 and AJ12631/pMWdAR6(EP0872547) or the like; L-methionine producing Escherichia bacteriasuch as AJ11539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJ11541 (NRRLB-12401), AJ 11542 (NRRL B-12402) (GB2075055) or the like; L-arginineproducing Escherichia bacteria such as AJ11531 and AJ11538(JP56106598A2), AJ11593 (FERM P-5616) and AJ11594 (FERM P-5617)(Japanese Patent Laid-open No. 57-5693) or the like.

The expression of one or more genes which encode proteins involved inthe biosynthesis of L-amino acids may also be increased or enhanced. InL-threonine producing bacteria, such genes include the genes of thethreonine operon, such as the gene encoding aspartate kinase, orhomoserine dehydrogenase which is desensitized to feedback inhibition byL-threonine (Japanese Patent Publication No. 1-29559). For L-valineproducing bacteria, such genes include the ilv operon, that is, theilvGMEDA operon, which does not preferably express threonine deaminaseand which has suppressed attenuation (Japanese Patent Laid-OpenPublication No. 8-47397). For L-proline producing bacteria, such genesinclude the proB gene encoding for glutamate kinase which has beendesensitized to feedback inhibition by L-proline (DE3127361). ForL-leucine producing bacteria, such genes include the leucine operon,that is, the leu operon, which preferably includes a gene coding forisopropylmalate synthase which is desensitized to feedback inhibition byL-leucine (Russian patent application 99114325). For L-methionineproducing bacteria, such genes include the methionine regulon. Themethionine regulon may have mutated genes coding for proteins which havea decreased activity for repressing amino acid biosynthesis. An exampleis the metJ gene coding for a L-methionine biosynthesis-relatingrepressor protein from E. coli, which has decreased activity forrepressing methionine biosynthesis (JP 2000-157267 A2). Furthermore,another example is the arginine regulon, which preferably includes agene encoding N-acetylglutamate synthase which is desensitized tofeedback inhibition by L-arginine (Rajagopal B. S. et al, Appl. Environ.Microbiol., 1998, v.64, No. 5, p. 1805-1811).

The method of the present invention includes a method for producingL-threonine, L-valine, L-proline, L-leucine, and/or L-methionine bycultivating the bacteria with enhanced activities of the proteins shownin SEQ ID NOs: 4 and 6 in a culture medium, allowing L-threonine toaccumulate in the culture medium, and collecting L-threonine from theculture medium.

The method of the present invention also includes a method for producingL-threonine and/or L-valine by cultivating the bacteria with enhancedactivity of the protein shown in SEQ ID NO: 12 in a culture medium,allowing L-threonine to accumulate in the culture medium, and collectingL-threonine from the culture medium.

The method of present invention further includes a method for producingL-arginine and/or L-proline by cultivating the bacteria with enhancedactivity of the protein shown in SEQ ID NO: 16 in a culture medium, toallow L-arginine to be produced and accumulated in the culture medium,and collecting L-arginine from the culture medium. Also, the method ofpresent invention includes a method for producing L-proline bycultivating the bacterium of the present invention in a culture medium,to allow L-proline to be produced and accumulated in the culture medium,and collecting L-proline from the culture medium.

The cultivation, collection, and purification of L-amino acids from themedium and the like may be performed in a manner similar to conventionalfermentation methods wherein an amino acid is produced using amicroorganism. The medium used for culture may be either synthetic ornatural, so long as it includes a carbon and nitrogen source, mineralsand, if necessary, appropriate amounts of nutrients required by thechosen microorganism for growth. The carbon source may include variouscarbohydrates such as glucose and sucrose, and various organic acids.Depending on the mode of assimilation of the chosen microorganism,alcohol including ethanol and glycerol may be used. As the nitrogensource, various ammonium salts such as ammonia and ammonium sulfate,other nitrogen compounds such as amines, a natural nitrogen source suchas peptone, soybean-hydrolysate and digested fermentative microorganismare used. As minerals, potassium monophosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride,and the like are used.

The cultivation is performed preferably under aerobic conditions such asa shaking culture, and stirring culture with aeration, at a temperatureof 20 to 40° C., preferably 30 to 38° C. The pH of the culture isusually between 5 and 9, preferably between 6.5 and 7.2. The pH of theculture can be adjusted with ammonia, calcium carbonate, various acids,various bases, and buffers. Usually, a 1 to 5-day cultivation leads tothe accumulation of the target L-amino acid in the liquid medium.

After cultivation, solids such as cells can be removed from the liquidmedium by centrifugation or membrane filtration, and then the targetL-amino acid can be collected and purified by ion-exchange,concentration and crystallization methods.

EXAMPLES

The present invention will be more concretely explained below withreference to the following non-limiting Examples. In the Examples, aminoacids are in the L-configuration unless otherwise noted.

Example 1 Cloning of the b2682, b2683, b1242, and b3434 Genes on thePlasmid pΔlacZ

To clone the b2682 and b2683 genes, vector pΔlacZ was used. pΔlacZ is aderivative of the pET-22b(+) vector (Novagen, Madison, Wis., USA).pET-22b(+) was treated by BglII and XbaI and ligated with the polymerasechain reaction (PCR) fragment of plasmid pMB9-lac (Fuller F., Gene, 19,43-54, 1982) which had been treated with the same restrictases andcarried the P_(lac) UV5 promoter. To amplify the P_(lac) UV5 promoterfragment by PCR, primers having the sequences depicted in SEQ ID Nos: 7and 8 were used. Then, the structural part of the lacZ gene (237 bpwithout promoter) was cloned into the plasmid using the SalI-BamHIfragment of the plasmid pJEL250 (Dymakova E. et al., Genetika (rus), 35,2, 181-186, 1999). The scheme for obtaining the pΔlacZ vector is shownin FIG. 1.

The PCR fragment obtained using DNA from E. coli strain TG1 as atemplate was used as the initial material to clone the E. coli b2682 andb2683 putative reading frames (b2682 and b2683 genes). The primershaving the sequences depicted in SEQ ID Nos: 1 and 2 were used tosynthesize this PCR fragment. PCR was carried out on “Perkin ElmerGeneAmp PCR System 2400” under the following conditions: 40 sec. at 95°C., 40 sec. at 47° C., 40 sec. at 72° C., 30 cycles. Thus, a 1158 bplinear DNA fragment containing the b2682 and b2683 genes was obtained.This PCR fragment was treated with the XbaI and BamHI restrictases andinserted into the multicopy vector pΔlacZ which had been previouslytreated with the same restrictases.

The plasmid with the PCR fragment was named pYGAZH, and carried both theb2682 and b2683 genes under the control of the lactose promoter (P_(lac)UV5).

Similarly, the PCR fragment obtained using DNA from E. coli strain TG1as a template was used as the initial material to clone the E. colib1242 putative reading frame (b1242 gene). The primers having thesequences depicted in SEQ ID Nos: 9 and 10 were used to synthesize thisfragment. The plasmid containing the PCR fragment was named pYCHE andcarried the b1242 gene under the control of the lactose promoter(P_(lac) UV5). The initial material for cloning of the E. coli b3434putative reading frame (b3434 gene) was the PCR fragment, which wasobtained using DNA from E. coli strain TG1 as a template. The primershaving the sequences depicted in SEQ ID Nos: 13 and 14 were used tosynthesize this fragment. The plasmid containing the PCR fragment wasnamed pYHGN and carried the b3434 gene under the control of the lactosepromoter (P_(lac) UV5).

Example 2 The Influence of the Amplified b2682 and b2683 Genes on theResistance of E. coli Strain TG1 to Amino Acids and Their Analogs

E. coli strains TG1(pYGAZH), TG1(pYCHE), and TG1(pYHGN), as well as acontrol TG1 strain having a vector without an insertion were grownovernight on LB medium supplemented with ampicillin (100 μg/ml). Thenight cultures of all the strains were diluted 25 times in fresh LBmedium supplemented with ampicillin (100 μg/ml) and IPTG (0.5 mM) andwere incubated 2 hours at 37° C. with aeration. The log phase cultureswere diluted in a 0.9% solution of NaCl and about 1000 cells were seededon plates with solid Adams medium supplemented with ampicillin (100μg/ml), IPTG (0.5 mM), and an amino acid or its analog. After 2-4 daysincubation at 37° C., the differences in colony size or colony numberbetween the TG1 cells with the hybrid plasmids and the control TG1 cellswere observed. The results of these experiments are presented in Table1.

TABLE 1 Concen- tration Effect on the growth of TG1 in strain havingplasmid Inhibitors media, μg/ml pYGAZH pYCHE pYHGN Proline 30000 No NoNo 3,4-Dihydroproline 23 R No No Isoleucine 18000 No No NoDL-Thiaisoleucine 1 R No No o-Methylthreonine 6 No No No L-Serine 2800No No No DL-Serine 3600 No No No DL-Serine hydroxamate 140 No No NoDL-o-Methylserine 3200 R R R 4-Azaleucine 45 R No No6-Diazo-5-oxo-L-norleucine 15 No No R Valine 7 R No No Methionine 38000No No No Norleucine 500 R No No Cysteine 1600 No No No Homoserine 1000No R No DL-β-Hydroxy-norvaline 80 No No R L-Aspartic acid β- 100 No NoNo hydroxamate Arginine 4300 No No No Lysine 5000 No No NoS-(2-Aminoethyl)cysteine 0.75 No No S Histidine 3000 No No NoL-Histidine hydroxamate 200 No No No DL-1,2,4-Triazole-3- 80 No No Noalanine Phenylalanine 13000 No No No p-Fluorophenylalanine 6 No No NoL-o-Fluorophenylalanine 1.7 R No No DL-o-Fluorophenylalanine 6 R No NoTryptophan 12500 No No No DL-4-Fluorotryptophan 0.1 No No No4-Methyltryptophan 0.25 No No No 7-Methyltryptophan 100 No No NoDL-a-Methyltryptophan 400 No No No m-Fluoro-DL-tyrosine 0.5 No No NoNo - no differences compared to the control strain R - more colonies orincreased colony size S - less colonies or decreased colony sizecompared to the control strain

Example 3 Production of Threonine by Cells Having Plasmid pYGAZH

The threonine producing strain VL2054 was transformed with the plasmidpYGAZH which contains the b2682 and b2683 genes under the control ofP_(lac) UV5 promoter. The resulting strain was named VL2054(pYGAZH). TheVL2054 strain is a derivative of the VKPM B-3996 strain and carries onits chromosome:

a) the integrated threonine operon under the control of the P_(R)promoter,

b) the wild-type rhtA gene,

c) the inactivated chromosomal gene encoding transhydrogenase (tdh gene)and the inactivated kanamycin resistant gene (kan) in the Tn5 (tdh::Tn5,Kan^(S)),

d) the mutation ilvA₄₄₂.

The VL2054 strain was deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) (Russia 113545, Moscow, 1 Dorozhnyproezd, 1) on Jan. 30, 2001 under accession number VKPM B-8067, andconverted to an international deposit based on the Budapest Treaty onFeb. 1, 2002.

5 colonies of each VL2054 strain, the control strain VL2054(pΔlacZ), andVL2054(pYGAZH) were suspended in 2 ml of minimal medium (11 g/l(NH₄)₂SO₄, 0.4 g/l NaCl, 0.4 g/l MgSO₄, 1 g/l K₂HPO₄, 1-mg/l FeSO₄, 10mg/l MnSO₄, 0.1 mg/l thiamin, 0.5 g/l yeast extract, 40 g/l glucose, 300mg/l ampicillin, if necessary) in 20-ml test tubes and incubatedovernight with aeration at 32° C. 0.2 ml of each night culture wastransferred to three 20-ml test tubes with 2 ml of fresh medium forfermentation with or without IPTG and cultivated at 32° C. for 48 or 72hours on a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 22 g/l NaCl 0.8 g/l MgSO₄ 0.8 g/l K₂HPO₄ 2 g/l FeSO₄ 20 mg/lMnSO₄ 20 mg/l Thiamin 0.2 mg/l Yeast extract 1 g/l CaCO₃ 30 g/l Glucose80 g/l Ampicillin 300 mg/l, if necessary IPTG 0.5 mM, if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofthreonine which accumulated in the medium was determined by thin layerchromatography (TLC). The liquid phase composition for TLC was asfollows: isopropanol (50 ml), acetone (50 ml), 30% NH₄OH (12 ml), H₂O (8ml). The results are shown in Table 2. As shown, the presence of thehybrid plasmid pYGAZH improved the amount of threonine accumulation bythe threonine producing strain VL2054.

TABLE 2 VL2054 48 hours 72 hours with Thr, Thr, plasmid IPTG OD₅₄₀ g/lThr/OD OD₅₄₀ g/l Thr/OD no − 19 5.2 0.27 26 9.1 0.35 + 21 4.1 0.20 297.8 0.27 pΔlacZ − 20 6.4 0.32 24 9.1 0.40 + 15 3.5 0.23 24 7.2 0.30pYGAZH − 17 5.7 0.34 24 9.7 0.40 + 21 9.8 0.47 23 15.5 0.67

Example 4 Production of Valine by a Strain with Plasmid pYGAZH

The valine producing strain H-81 was transformed with the pYGAZH plasmidwhich contains the b2682 and b2683 genes under the control of P_(lac)UV5 promoter. The H-81 strain was deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) (Russia 113545, Moscow, 1Dorozhny proezd, 1) on Jan. 30, 2001 under accession number VKPM B-8066,and converted to an international deposit based on the Budapest Treatyon Feb. 1, 2002.

5 colonies of each H-81 strain, control strain H-81(pΔlacZ), andH-81(pYGAZH) were suspended in 2 ml of minimal medium (18 g/l (NH₄)₂SO₄,1.8 g/l K₂HPO₄, 1.2 g/l MgSO₄, 0.1 g/l thiamin, 0.5 g/l yeast extract,60 g/l glucose, 100 mg/l ampicillin, if necessary), in 20-ml test tubesand incubated overnight with aeration at 32° C. 0.2 ml of each nightculture was transferred to three 20-ml test tubes with 2 ml of freshmedium for fermentation with or without IPTG and cultivated at 32° C.for 48 or 72 hours on a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 18 g/l, K₂HPO₄ 1.8 g/l, MgSO₄ 1.2 g/l, CaCO₃ 20 g/l, Thiamin0.1 mg/l, Glucose 60 g/l, Ampicillin 300 mg/l, if necessary IPTG 0.5 mM,if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofvaline which accumulated in the medium was determined by TLC. The liquidphase composition for TLC was as follows: isopropanol (80 ml),ethylacetate (80 ml), 30% NH₄OH (15 ml), H₂O (45 ml). The results areshown in Table 3. As shown, the presence of the hybrid plasmid pYGAZHimproved the amount of valine which accumulated by the valine producingstrain H-81.

TABLE 3 48 hours 72 hours H-81 with Val/ Val/ plasmid IPTG OD₅₄₀ Val,g/l OD OD₅₄₀ Val, g/l OD No − 34 11.6 0.34 32 10.3 0.32 + 34 11.7 0.3430 10.1 0.34 pΔlacZ − 34 10.5 0.31 30 10.0 0.33 + 20 7.8 0.39 25 9.00.36 pYGAZH − 29 10.5 0.36 31 12.8 0.41 + 22 10.8 0.49 23 12.3 0.53

Reference Example 1 Production of L-proline by an ilvA DeficientL-proline Producer

Cells of wild-type strain E. coli K12 (VKPM B-7) were treated with themutagen N-methyl-N′-nitro-N-nitrosoguanidine (0.1 mg/ml) for 20 min at37° C., washed and plated on minimal agar medium M9 supplemented with1.25 mg/ml tryptone, 10 mg/ml L-proline, and 0.05 mg/ml2,3,5-triphenyltetrazolium chloride. Most colonies which appeared after3 days of incubation at 37° C. were red. A few colonies which could notoxidize L-proline were white. One of these colonies was used as a parentto obtain mutants resistant to proline analogs (3,4-dehydroxyproline andazetidine-2-carboxylate), which were added to M9 agar medium to aconcentration of 2 mg/ml each.

Some of mutants which appeared could produce L-proline. The bestL-proline producer, 702, was treated with a P1 bacteriophage grown oncells of the TG1 strain in which the ilvA gene was disrupted by theinsertion of the chloramphenicol (Cm) resistance (Cm^(r)) gene. One ofCm resistant transductants, 702ilvA, which turned out to be L-isoleucineauxotrophic, was much more effective at producing L-proline than theL-isoleucine prototrophic parent strain 702 (Table 4). The fermentationmedium contained 60 g/l glucose, 25 g/l ammonium sulfate, 2 g/l KH₂PO₄,1 g/l MgSO₄, 0.1 mg/l thiamine, 50 mg/l L-isoleucine and 25 g/l chalk(pH 7.2). Glucose and chalk were sterilized separately. 2 ml of themedium was placed into test tubes, and inoculated with one loop of thetested microorganisms, and the cultivation was carried out at 37° C. for2 days with shaking.

TABLE 4 Accumulation of L- Strain Phenotype proline (g/l) K12 (VKPM B-7)Wild-type <0.1 702 (VKPM B-8011) Defective L-proline 0.5 degradation,resistance to proline analogs 702ilvA (VKPM B- Defective L-proline 8.08012) degradation, resistance to proline analogs, L-isoleucineauxotroph, Cm^(r)

The 702 and 702ilvA strains were deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) under the accessionnumber VKPM B-8011 and VKPM B-8012, respectively, on Jul. 25, 2000.

Example 5 Production of Proline by a Strain Having Plasmid pYGAZH

The proline producing strain E. coli 702ilvA was transformed with theplasmid pYGAZH which contains the b2682 and b2683 genes under thecontrol of P_(lac) UV5 promoter.

5 colonies of each 702ilvA strain, control strain 702ilvA(pΔlacZ), and702ilvA(pYGAZH) were suspended in 2 ml of minimal medium (18 g/l(NH₄)₂SO₄, 1.8 g/l K₂HPO₄, 1.2 g/l MgSO₄, 0.1 mg/l thiamin, 0.5 g/lyeast extract, 60 g/l glucose, 50 mg/l isoleucine, 300 mg/l ampicillin,if necessary) in 20-ml test tubes and incubated overnight with aerationat 32° C. 0.2 ml of each night culture was transferred to three 20-mltest tubes with 2 ml of fresh medium for fermentation with or withoutIPTG and cultivated at 32° C. for 40 hours with rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 18 g/l, K₂HPO₄ 1.8 g/l, MgSO₄ 1.2 g/l, CaCO₃ 20 g/l, Thiamin0.1 mg/l, Glucose 60 g/l, Isoleucine 50 mg/l Ampicillin 300 mg/l, ifnecessary IPTG 0.5 mM, if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofproline which accumulated in the medium was determined by TLC. Theliquid phase composition for TLC was as follows: ethanol (80 ml), 30%NH₄OH (5 ml), H₂O (25 ml). The results are shown in Table 5. As shown,the presence of the hybrid plasmid pYGAZH improved the accumulation ofproline by the proline producing strain 702ilvA.

TABLE 5 702ilvA 40 hours with plasmid IPTG OD₅₄₀ Pro, g/l Pro/OD No − 254.0 0.16 + 23 4.1 0.18 pΔlacZ − 24 5.3 0.22 + 22 5.0 0.23 pYGAZH − 215.0 0.24 + 23 10.6 0.46

Reference Example 2 Production of L-Leucine by an ilvE DeficientL-Leucine Producer

Cells of wild-type strain E. coli K12 (VKPM B-7) were treated with themutagen N-methyl-N′-nitro-N-nitrosoguanidine (0.05 mg/ml) for 20 min at37° C., washed 4 times with physiological solution and plated on minimalagar medium M9 supplemented with 4.0 mg/ml DL-4-azaleucine. The plateswere incubated for 5 days at 37° C. Colonies which appeared on theplates were picked up and purified by streaking on the L-agar plates.One of the mutants which showed resistance to DL-4-azaleucine was usedto induce double L-isoleucine and L-valine auxotrophy. Numerous doubleauxotrophs which require L-isoleucine and L-valine for growth wereobtained. Double L-isoleucine and L-valine auxotrophy was shown to becaused by a mutation in the ilvE gene. The best L-leucine producer ofthe double auxotrophs was selected and shown to be strain 505, whichproduced 1.8 g/l of L-leucine. The fermentation medium contained 60 g/lglucose, 25 g/l ammonium sulfate, 2 g/l KH₂PO₄, 1 g/l MgSO₄, 0.1 mg/lthiamine, 100 mg/l L-isoleucine, 100 mg/l L-valine and 25 g/l chalk (pH7.2). Glucose and chalk were sterilized separately. 2 ml of the mediumwas placed into test tubes, and inoculated with one loop of the testedmicroorganisms, and the cultivation was carried out at 37° C. for 2 dayswith shaking.

The E. coli 505 strain was deposited in the Russian National Collectionof Industrial Microorganisms (VKPM) (Russia 113545, Moscow, 1 Dorozhnyproezd, 1) on May 14, 2001 under accession number VKPM B-8124, andconverted to an international deposit under the Budapest Treaty on Feb.1, 2002.

Example 6 Production of Leucine by a Strain Having Plasmid pYGAZH

The leucine producing strain E. coli 505 was transformed by the plasmidpYGAZH which contained the b2682 and b2683 genes under the control ofthe P_(lac) UV5 promoter.

20 colonies of each strain 505, control strain 505(pΔlacZ), and505(pYGAZH) were transferred by one loop of culture to 20-ml test tubeswith L-broth with or without ampicillin, and were incubated overnightwith aeration at 32° C. 0.1 ml of each night culture was transferredinto the 20-ml test tubes (inner diameter 22 mm), suspended in 2 ml ofmedium for fermentation with or without IPTG, and cultivated at 32° C.for 72 hours on a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 15 g/l, K₂HPO₄ 1.5 g/l, MgSO₄ × 7H₂O 1.0 g/l, CaCO₃ 20 g/l(sterilized separately), Thiamin 0.1 mg/l, Glucose 60 g/l (sterilizedseparately), Isoleucine 0.3 g/l Valine 0.3 g/l Ampicillin 150 mg/l, ifnecessary IPTG 0.5 mM, if necessary

After cultivation, the plasmid stability was determined by aconventional method. The amount of leucine which accumulated in themedium was determined by TLC. The liquid phase composition for TLC wasas follows: isopropanol (80 ml), ethylacetate (80 ml), 30% NH₄OH (25ml), H₂O (0 ml). The results are shown in Table 6. As shown, thepresence of the hybrid plasmid pYGAZH improved the accumulation ofleucine by the leucine producing 505 strain.

TABLE 6 505 72 hours with plasmid IPTG Leu, g/l No − 1.8 + 2.0 pΔlacZ −1.8 + 2.0 pYGAZH − 2.0 + 2.8

Reference Example 3 Production of L-Methionine by L-Methionine ProducerResistant to Norleucine

The plasmidless threonine and leucine deficient E. coli C600 strain wasused to derive the following strains. At first, Leu⁺ variants of E. coliC600 strain were obtained by transduction of phage P1 grown on E. coliK-12 strain. Then, after treatment withN-methyl-N′-nitro-N-nitrosoguanidine (NTG), the mutant strain 44 wasobtained, which is resistant to 8 g/l of L-homoserine. The 44 strain isL-threonine-deficient and resistant to high concentrations ofL-homoserine. The 44 strain was deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) under the accessionnumber VKPM B-2175.

Then, mutant strains resistant to the methionine analog norleucine wereinduced from the 44 strain by mutagenesis using NTG. The cells of thenight culture grown in L-broth were spun down and resuspended inphysiological solution (0.9% NaCl) containing 50 μg/ml of NTG. After 30min of exposure with NTG at 37° C., the cells were spun down, washed 4times with physiological solution, and plated on the minimal agar mediumM9 containing 0.5 mg/ml of threonine and 2.5 mg/ml or 5.0 mg/ml ofnorleucine. The plates were incubated for 5 days at 37° C. Colonieswhich appeared on the plates were purified by streaking on L-agarplates. Strain 218 turned out to be the best L-methionine producer.Test-tube cultivation of the novel 218 strain was performed at 32° C.for 3 days with shaking, and resulted in accumulation in the culturemedium of about 1 g/l L-methionine. A fermentation medium of minimalmedium M9 containing glucose (4%), ammonia sulfate (2.5%), threonine(0.5 g/l), calcium carbonate (25 g/l) was used. Glucose and chalk weresterilized separately.

The 218 strain was deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) under the accession number VKPM B-8125on May 14, 2001, and converted to an international deposit under theBudapest Treaty on Feb. 1, 2002.

Furthermore, the ppc gene was deleted via P1 phage in the 218 strain,followed by integration of the pycA gene from Bacillus subtilis (Russianpatent application 99121636). The resulting strain 218pycA lost itsresistance to norleucine. Therefore, resistance to norleucine wasimparted to the strain again as described above. The best L-methionineproducer among obtained strains was strain E. coli 73 which producedabout 1 g/l of L-methionine under the conditions described above.

The E. coli 73 strain was deposited in the Russian National Collectionof Industrial Microorganisms (VKPM) (Russia 113545 Moscow 1 Dorozhnyproezd, 1) on May 14, 2001 under accession number VKPM B-8126, andconverted to an international deposit under the Budapest Treaty on Feb.1, 2002.

Example 7 Production of Methionine by a Strain Having Plasmid pYGAZH

The methionine producing E. coli 73 strain was transformed with theplasmid pYGAZH which contains the b2682 and b2683 genes under thecontrol of P_(lac) UV5 promoter.

5 colonies of each strain 73, control stain 73(pΔlacZ), and 73(pYGAZH)were suspended in 2 ml of minimal medium (18 g/l (NH₄)₂SO₄, 1.8 g/lK₂HPO₄, 1.2 g/l MgSO₄, 0.1 mg/l thiamin, 10 g/l yeast extract, 60 g/lglucose, 400 mg/l threonine, 300 mg/l ampicillin, if necessary) in 20-mltest tubes and incubated overnight with aeration at 32° C. 0.2 ml ofeach night culture was transferred to three 20-ml test tubes with 2 mlof fresh medium for fermentation with or without IPTG, and cultivated at32° C. for 48 hours on a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 18 g/l, K₂HPO₄ 1.8 g/l, MgSO₄ 1.2 g/l, CaCO₃ 20 g/l, Thiamin0.1 mg/l, Glucose 60 g/l, Threonine 400 mg/l, Yeast extract 1.0 g/l,Ampicillin 300 mg/l, if necessary IPTG 0.5 mM, if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofmethionine which accumulated in the medium was determined by TLC. Theliquid phase composition for TLC was as follows: isopropanol (80 ml),ethylacetate (80 ml), 30% NH₄OH—(15 ml), H₂O (45 ml). The results areshown in Table 7. As shown, the presence of the hybrid plasmid pYGAZHimproved the methionine accumulation by the methionine producing 73strain.

TABLE 7 48 hours 73 with plasmid IPTG OD₅₄₀ Met, g/l Met/OD No − 45 0.70.016 + 42 1.1 0.026 pΔlacZ − 45 1.0 0.022 pYGAZH − 48 0.9 0.019 + 461.3 0.028

Example 8 Production of Threonine by a Strain Having Plasmid pYCHE

The threonine producing VL2054 strain was transformed with the plasmidpYCHE containing the b1242 gene under the control of P_(lac) UV5promoter. The resulting strain was named VL2054(pYCHE).

5 colonies of each strain VL2054, control strain VL2054(pΔlacZ), andVL2054(pYCHE) were suspended in 2 ml of minimal medium (11 g/l(NH₄)₂SO₄, 0.4 g/l NaCl, 0.4 g/l MgSO₄, 1 g/l K₂HPO₄, 10 mg/l FeSO₄, 10mg/l MnSO₄, 0.1 mg/l thiamin, 0.5 g/l yeast extract, 40 g/l glucose, 300mg/l ampicillin, if necessary) in 20-ml test tubes and incubatedovernight with aeration at 32° C. 0.2 ml of each night culture wastransferred to three 20-ml test tubes with 2 ml of fresh medium forfermentation with or without IPTG, and cultivated at 32° C. for 45 hourson a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 22 g/l NaCl 0.8 g/l MgSO₄ 0.8 g/l K₂HPO₄ 2 g/l FeSO₄ 20 mg/lMnSO₄ 20 mg/l Thiamin 0.2 mg/l Yeast extract 1 g/l CaCO₃ 30 g/l Glucose80 g/l Ampicilline 300 mg/l, if necessary IPTG 0.5 mM, if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofthreonine which accumulated in the medium was determined by thin layerchromatography (TLC). The liquid phase composition for TLC was asfollows: isopropanol (50 ml), acetone (50 ml), 30% NH₄OH (12 ml), H₂O (8ml). The results are shown in Table 8. As shown, the presence of thehybrid plasmid pYCHE improved the threonine accumulation by thethreonine producing strain VL2054.

TABLE 8 VL2054 with plasmid IPTG OD₅₄₀ Thr, g/l Thr/OD no − 21 4.80.23 + 20 4.7 0.24 pΔlacZ − 16 4.6 0.29 + 13 3.0 0.23 pYCHE − 20 6.20.31 + 20 7.0 0.35

Example 9 Production of Valine by a Strain Having Plasmid pYCHE

The valine producing strain H-81 was transformed with the plasmid pYCHEwhich contains the b1242 gene under the control of P_(lac) UV5 promoter.

5 colonies of each strain H-81, control strain H-81(pΔlacZ), andH-81(pYCHE) were suspended in 2 ml of minimal medium (18 g/l (NH₄)₂SO₄,1.8 g/l K₂HPO₄, 1.2 g/l MgSO₄, 0.1 mg/l thiamin, 0.5 g/l yeast extract,0 g/l glucose, 300 mg/l ampicillin, if necessary) in 20-ml test tubesand incubated overnight with aeration at 32° C. 0.2 ml of each nightculture was transferred to three 20-ml test tubes with 2 ml of freshmedium for fermentation with or without IPTG, and cultivated at 32° C.for 45 hours on a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 18 g/l, K₂HPO₄ 1.8 g/l, MgSO₄ 1.2 g/l, CaCO₃ 20 g/l, Thiamin0.1 mg/l, Glucose 60 g/l, Ampicilline 300 mg/l, if necessary IPTG 0.5mM, if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofvaline which accumulated in the medium was determined by TLC. The liquidphase composition for TLC was as follows: 80 ml isopropanol, 80 mlethylacetate, 15 ml 30% NH₄OH, 45 ml H₂O. The results are shown in Table9. As shown, the presence of the hybrid plasmid pYCHE improved thevaline accumulation by the valine producing strain H-81.

TABLE 9 H-81 with plasmid IPTG OD₅₄₀ Val, g/l Val/OD no − 34 11.6 0.34 +34 11.7 0.34 pΔlacZ − 34 10.5 0.31 + 20 7.8 0.39 pYCHE − 32 14.0 0.44 +30 13.9 0.46

Example 10 Production of Arginine by a Strain Having Plasmid pYHGN

The arginine producing strain 382 was transformed with the plasmid pYHGNwhich contains the b3434 gene under the control of the P_(lac) UV5promoter. The 382 strain was deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) (Russia 113545, Moscow, 1Dorozhny proezd, 1) on Apr. 10, 2000 under accession number VKPM B-7926.

5 colonies of each strain 382, control strain 382(pΔlacZ), and382(pYHGN) were suspended in 2 ml of minimal medium (25.0 g/l (NH₄)₂SO₄,2.0 g/l K₂HPO₄, 1.0 g/l MgSO₄ 7H₂O, 0.1 mg/l thiamin, 5 g/l yeastextract, 60 g/l glucose, 100 mg/l ampicillin, if necessary) in 20-mltest tubes and incubated overnight with aeration at 32° C. 0.2 ml ofeach night culture was transferred to three 20-ml test tubes with 2 mlof fresh medium for fermentation with or without IPTG, and cultivated at32° C. for 72 hours on a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 25 g/l, K₂HPO₄ 2.0 g/l, MgSO₄7H₂O 1.0 g/l, Thiamin 0.2 mg/l,Yeast extract 5 g/l Glucose 60 g/l, CaCO₃ 20 g/l Ampicilline 100 mg/l,if necessary IPTG 0.5 mM, if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofarginine which accumulated in the medium was determined by TLC. Theliquid phase composition for TLC was as follows: 80 ml isopropanol, 40ml ethylacetate, 15 ml 30% NH₄OH, 50 ml H₂O. The results are shown inTable 10. As shown, the presence of the hybrid plasmid pYHGN improvedthe arginine accumulation by the arginine producing strain 382.

TABLE 10 E. coli 382 with plasmid IPTG OD₅₄₀ Arg, g/l Arg/OD No − 20 8.50.43 + 22 6.7 0.31 pΔlacZ − 28 6.3 0.23 + 26 5.4 0.21 pYHGN − 24 5.80.24 + 26 9.3 0.36

Example 11 Production of Proline by a Strain Having Plasmid pYHGN

The proline producing strain E. coli 702ilvA was transformed with theplasmid pYHGN which contains the b3434 gene under the control of theP_(lac) UV5 promoter.

5 colonies of each strain 702ilvA, control strain 702ilvA(pΔlacZ), and702ilvA(pYHGN) were suspended in 2 ml of minimal medium (18 g/l(NH₄)₂SO₄, 1.8 g/l K₂HPO₄, 1.2 g/l MgSO₄, 0.1 mg/l thiamin, 0.5 g/lyeast extract, 60 g/l glucose, 50 mg/l isoleucine, 300 mg/l ampicillin,if necessary) in 20-ml test tubes and incubated overnight with aerationat 32° C. 0.2 ml of each night culture was transferred to three 20-mltest tubes with 2 ml of fresh medium for fermentation with or withoutIPTG, and cultivated at 32° C. for 40 hours on a rotary shaker.

Fermentation Medium Composition:

(NH₄)₂SO₄ 18 g/l, K₂HPO₄ 1.8 g/l, MgSO₄ 1.2 g/l, CaCO₃ 20 g/l, Thiamin0.1 mg/l, Glucose 60 g/l, Isoleucine 50 mg/l Ampicilline 300 mg/l, ifnecessary IPTG 0.5 mM, if necessary

After cultivation, the plasmid stability and optical absorbance of themedium at 540 nm were determined by conventional methods. The amount ofproline which accumulated in the medium was determined by TLC. Theliquid phase composition for TLC was as follows: 80 ml ethanol, 5 ml 30%NH₄OH, 25 ml H₂O. The results are shown in Table 11. As shown, thepresence of the hybrid plasmid pYHGN improved the proline accumulationby the proline producing strain 702ilvA.

TABLE 11 40 hours 702ilvA Pro/ with plasmid IPTG OD₅₄₀ Pro, g/l OD No −25 4.0 0.16 + 23 4.1 0.18 PΔlacZ − 24 5.3 0.22 + 22 5.0 0.23 pYHGN − 245.9 0.25 + 17 7.1 0.42

Modifications and Other Embodiments

Various modification and variations of the described products,compositions, and methods as well as the concept of the invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed is not intended to be limitedto such specific embodiments. Various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the biochemical, chemical, chemical engineering, molecularbiological, medical, or pharmacological arts or related fields areintended to be within the scope of the following claims.

INCORPORATION BY REFERENCE

Each document, patent application or patent publication cited by orreferred to in this disclosure is incorporated by reference in itsentirety. Any patent document to which this application claims priorityis also incorporated by reference in its entirety. Specifically,priority documents Russian Patent Application No. 2001103865, filed Feb.13, 2001; Russian Patent Application No. 2001104998, filed Feb. 26,2001; Russian Patent Application No. 2001104999, filed Feb. 26, 2001;Russian Patent Application 2001117632, filed Jun. 28, 2001; and RussianPatent Application No. 2001117633, filed Jun. 28, 2001 are herebyincorporated by reference.

1. An isolated L-amino acid producing Escherichia bacterium withincreased expression of a gene encoding a protein selected from thegroup consisting of: (A) a protein comprising the amino acid sequence inSEQ ID NO: 12; and (B) a protein comprising the amino acid sequence ofSEQ ID NO: 12 except that a total of between 1 and 5 amino acids aredeleted, substituted, inserted, or added, wherein the expression of saidprotein is increased by transformation of said bacterium with the genecoding for said protein, or by placing said gene under the control of apotent promoter.
 2. The bacterium according to the claim 1, wherein thetransformation is performed with a multicopy vector.
 3. The bacteriumaccording to claim 1, wherein the protein (A) is encoded by thepolynucleotide which has the nucleotide sequence of SEQ ID NO:
 11. 4.The bacterium according to claim 1, wherein the protein (B) is encodedby a polynucleotide which hybridizes with a sequence which iscomplementary to the nucleotide sequence of SEQ ID NO: 11 underconditions comprising washing in 1×SSC and 0.1% SDS at 60° C.
 5. Amethod for producing an L-amino acid comprising: A) cultivating thebacterium according to claim 1 in a culture medium, and B) collectingthe L-amino acid from the culture medium and/or bacterium.
 6. The methodaccording to claim 5, wherein the L-amino acid is L-threonine.
 7. Themethod according to claim 6, wherein the bacterium has been modified tohave enhanced expression of the threonine operon as compared to anon-modified bacterium.
 8. The method according to claim 5, wherein theL-amino acid is L-valine.
 9. The method according to claim 8, whereinthe bacterium has been modified to have enhanced expression of the ilvoperon as compared to a non-modified bacterium.
 10. A method forproducing an L-amino acid comprising: A) cultivating the bacteriumaccording to claim 2 in a culture medium, and B) collecting the L-aminoacid from the culture medium and/or bacterium.
 11. The method accordingto claim 10, wherein the L-amino acid is L-threonine.
 12. The methodaccording to claim 11, wherein the bacterium has been modified to haveenhanced expression of the threonine operon as compared to anon-modified bacterium.
 13. The method according to claim 10, whereinthe L-amino acid is L-valine.
 14. The method according to claim 13,wherein the bacterium has been modified to have enhanced expression ofthe ilv operon as compared to a non-modified bacterium.